Qualitative Analysis of Amino Acid

.
.
Theory
Procedure
Self Evaluation
Animation
Simulator
Assignment
Reference
Feedback

Go to simulator
b!ectives"

To detect the #resence of amino acid from a given unkno$n sam#le.

Theory"



Amino acids are building blocks of all #roteins% and are linked in series by
#e#tide bond &'()*'+ to form the #rimary structure of a #rotein. Amino
acids #ossess an amine grou#% a carbo,ylic acid grou# and a varying side
chain that di-ers bet$een di-erent amino acids.
There are ./ naturally occurring amino acids% $hich vary from one another
$ith res#ect to their side chains. Their melting #oints are e,tremely high
&usually e,ceeding .//0(+% and at their #1% they e,ist as 2$itterions% rather
than as unioni2ed molecules.
Amino acids res#ond to all ty#ical chemical reactions associated $ith
com#ounds that contain carbo,ylic acid and amino grou#s% usually under
conditions $here the 2$itter ions form is #resent in only small 3uantities. All
amino acids &e,ce#t glycine+ e,hibit o#tical activity due to the #resence of an
asymmetric 4 5 (arbon atom. Amino acids $ith an 6 5 configuration are
#resent in all naturally occurring #roteins% $hereas those $ith 7 5 forms are
found in antibiotics and in bacterial cell $alls.




Fig 8. Structure of amino acid

Princi#le"

)inhydrin test

1n the #* range of 9':% all 4' amino acids react $ith ninhydrin
&triketohydrindene hydrate+% a #o$erful o,idi2ing agent to give a #ur#le
colored #roduct &diketohydrin+ termed Rhuemann;s #ur#le. All #rimary
amines and ammonia react similarly but $ithout the liberation of carbon
dio,ide. The imino acids #roline and hydro,y#roline also react $ith ninhydrin%
but they give a yello$ colored com#le, instead of a #ur#le one. <esides
amino acids% other com#le, structures such as #e#tides% #e#tones and
#roteins also react #ositively $hen sub!ected to the ninhydrin reaction.

=antho#roteic acid test
Aromatic amino acids% such as Phenyl alanine% tyrosine and try#to#han%
res#ond to this test. 1n the #resence of concentrated nitric acid% the aromatic
#henyl ring is nitrated to give yello$ colored nitro'derivatives. At alkaline #*%
the color changes to orange due to the ioni2ation of the #henolic grou#.

Pauly>s dia2o Test
This test is s#eci?c for the detection of Try#to#han or *istidine. The reagent
used for this test contains sul#hanilic acid dissolved in hydrochloric acid.
Sul#hanilic acid u#on dia2oti2ation in the #resence of sodium nitrite and
hydrochloric acid results in the formation a dia2onium salt. The dia2onium
salt formed cou#les $ith either tyrosine or histidine in alkaline medium to
give a red coloured chromogen &a2o dye+.
@illon>s test
Phenolic amino acids such as Tyrosine and its derivatives res#ond to this test.
(om#ounds $ith a hydro,yben2ene radical react $ith @illon;s reagent to form
a red colored com#le,. @illon;s reagent is a solution of mercuric sul#hate in
sul#huric acid.
*istidine test

This test $as discovered by Anoo#. This reaction involves bromination of
histidine in acid solution% follo$ed by neutrali2ation of the acid $ith e,cess of
ammonia. *eating of alkaline solution develo#s a blue or violet coloration.
*o#kins cole test
This test is s#eci?c test for detecting try#to#han. The indole moiety of
try#to#han reacts $ith glyo,ilic acid in the #resence of concentrated
sul#huric acid to give a #ur#le colored #roduct. Glyo,ilic acid is #re#ared from
glacial acetic acid by being e,#osed to sunlight.

Sakaguchi test

Bnder alkaline condition% 4' na#hthol &8'hydro,y na#hthalene+ reacts $ith a
mono'substituted guanidine com#ound like arginine% $hich u#on treatment
$ith hy#obromite or hy#ochlorite% #roduces a characteristic red color.

6ead sul#hide test

Sul#hur containing amino acids% such as cysteine and cystine. u#on boiling
$ith sodium hydro,ide &hot alkali+% yield sodium sul#hide. This reaction is due
to #artial conversion of the organic sul#hur to inorganic sul#hide% $hich can
detected by #reci#itating it to lead sul#hide% using lead acetate solution.



Folin>s @c(arthy Sullivan Test
1mino acids such as Proline and hydro,y#roline condense $ith isatin reagent
under alkaline condition to yield blue colored adduct. Addition to sodium
nitro#russideC)a.Fe&()+D)E to an alkaline solution of methionine follo$ed
by the acidi?cation of the reaction yields a red colour. This reaction also forms
the basis for the 3uantitative determination of methionine.

1satin test

1mino acids such as Proline and hydro,y#roline condense $ith isatin reagent
under alkaline condition to yield blue colored adduct.